Peptides, which exhibit biological activity for the first time when C-terminal is amidated, for example, calcitonin, gastrin, secretin, vasoactive intestinal polypeptide, growth hormone-releasing factor, corticotropin-releasing factor, etc. have been known to be formed from glycine adducts through enzymatic reactions in vivo. Many of these biologically active peptides are useful as pharmaceuticals, and presently calcitonin, secretin, among others, are commercially available as pharmaceuticals.
These peptides have been obtained primarily by separation and purification from living bodies, but the steps are cumbersome and living bodies as sources are obtained only with difficulty. Therefore, the above presently commercially available peptides are very expensive.
Accordingly, in recent years, attempts have been made to produce these biologically active peptides by using a recombinant DNA technique. But according to the recombinant DNA technique using Escherichia coli, yeast, Bacillus subtilis, and the like as the host, an C-terminal amidation of the peptide produced cannot be effected, which has been an obstacle to producing the above peptide. Accordingly, there is a demand for a technique whereby C-terminal amidation is effected easily and inexpensively in vitro.
The enzyme participating in such amidation is called peptidylglycine-.alpha.-amidating monoxygenase (C-terminal amidating enzyme) (EC.1.14.17.3) (Bradbury et al, Nature, 298, 686, 1982: Glembotski et al, J. Biol, Chem., 259, 6385, 1984),and is considered to catalyze the following reaction: EQU --CHCONHCH.sub.2 COOH.fwdarw.--CHCONH.sub.2 +glyoxylic acid
To clarify the amidation mechanism in vivo and amidate the peptide produced by the recombinant technique at the C-terminal in vitro, attempts have been made to purify this enzyme. As examples which have a purified specific activity to 100-fold based source or higher, there have been reported those derived from bovine pituitary gland middle lobe (Murthy et al, J. Biol. Chem., 261, 1815, 1986), porcine pituitary gland (Kizer et al, Endocrinology, 118, 2262, 1986; Bradbury et al, Eur. J. Blochem., 169, 579, 1987), porcine heart atrium (Kojima et al, J. Blochem., 105, 440, 1989), Xenopus body skin (Mizuno et al, Biochem., Biophys. Res. Commun., 137, 984, 1986, rat thyroid gland tumor (Mehta et al, Arch. Biochem., Biophys., 261, 44, 1988). But, except for the method of Kizer et al, the operation is cumbersome, involving 5 to 6 purification steps. Also, according to the method of Kizer et al, there is the step of Sephadex G-100 (produced by Pharmacia) gel filtration, and this takes a long time for elution, and at the same time it is difficult to treat a large amount of the product.
In addition, concerning the existence of the C-terminal amidating enzyme in blood, there are reports of rat (Eipper et al, Endocrinology, 116, 2497, 1985) and human (Wand et al, Metabolism, 34, 1044, 1985), but both have low specific activities and no attempt at purification thereof have been made.
As described above, no purification method capable of simple and bulk treatment of the C-terminal amidating enzyme has been established. Also, no purification has been done from serum and plasma, and no method of producing a peptide amidated at the C-terminal or derivative thereof at low cost and in large which utilizing a C-terminal amidating enzyme in vitro, is known.